Project Summary/Abstract The human immune system produces at least 140 different antimicrobial peptides (AMPs) to kill invading bacteria. However, pathogenic bacteria use specialized pathways called two-component systems (TCSs) to detect these AMPs and activate the expression of AMP-resistance and virulence genes. This response enables pathogens to survive immune attacks and mount deadly infections. Therefore, elucidating the mechanisms by which peptides interact with TCSs is critical to understanding how infections progress. This knowledge could also lead to the design of new antimicrobial drugs that interfere with TCS-mediated AMP sensing. Gram-negative Enterobacteriaceae, such as the common pathogen Salmonella Typhimurium, cause 200,000 infections and 10,000 deaths in the United States each year. The most important AMP-sensing TCS in Gram-negative Enterobacteriaceae is named PhoPQ. Here, the membrane bound histidine kinase PhoQ senses AMPs and responds by phosphorylating the cytoplasmic response regulator PhoP, which activates a gene expression response. Though its interactions with a small number of model AMPs have been characterized, little is known about the broader peptide binding and sensing capabilities of PhoQ. The major limitations have been the cost and time required to chemically synthesize peptides and characterize their effects on TCSs using traditional microbiological or biochemical methods. In preliminary work, we have developed a new technology named SLAY-TCS that combines bacterial peptide display, fluorescence-activated cell sorting, and next-generation DNA sequencing to measure how S. Typhimurium PhoQ responds to millions of peptides in a single experiment. Using SLAY-TCS, we have already revealed that PhoQ senses a far wider range of peptides than previously known. Here, we propose to use SLAY-TCS to characterize how S. Typhimurium PhoQ responds to nearly every AMP produced by the human immune system, and thousands of mutants thereof, in order to reveal the identities, sequence motifs, and biophysical properties of PhoQ-activating peptides (Aim 1). We will also combine this approach with PhoQ mutational analyses to reveal how PhoQ sensing specificity has evolved across diverse pathogens, which may have enabled them to adapt to different biogeographical locations in vivo (Aim 2). Finally, we will use SLAY-TCS to perform the first large-scale characterization of peptide inhibitors of PhoQ, and explore the efficacy of the strongest inhibitors we identify in preventing S. Typhimurium virulence in primary mouse macrophages (Aim 3). The work in Aim 3 will reveal mechanisms by which exogenously-delivered peptides can inhibit PhoQ, and could lead to the design of novel antimicrobial therapeutics based on modified peptides in the future. Taken together, this proposal will substantially enhance our understanding of how a dangerous family of bacteria causes infections in humans and accelerate the design of sorely-needed antimicrobial ...